CN106164663A - Conductive hydrogel for affine sensing - Google Patents

Conductive hydrogel for affine sensing Download PDF

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Publication number
CN106164663A
CN106164663A CN201480069974.3A CN201480069974A CN106164663A CN 106164663 A CN106164663 A CN 106164663A CN 201480069974 A CN201480069974 A CN 201480069974A CN 106164663 A CN106164663 A CN 106164663A
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bio
electrode
hydrogel
pedot
identification part
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A·瑞夫辛
Z·马萨卢
俞定穆
权恩真
刘颖
申东植
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University of California
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University of California
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
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    • G01N33/5438—Electrodes
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    • C08L65/00—Compositions of macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain; Compositions of derivatives of such polymers
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/002—Electrode membranes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
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    • C12Q1/003—Functionalisation
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    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Abstract

The invention provides a kind of sensor for detecting disease marker, and the method for detection disease marker, described sensor and method make use of the conductive hydrogel that bio-identification part is modified.In one embodiment, the invention provides a kind of sensor, it has base plate；At least one contacts the electrode of described base plate；Cover the nano-porous films of described electrode；And selected from the bio-identification part of lower group: peptide, antibody, enzyme and fit, wherein said bio-identification some covalent is attached to electrode, or is covalently bound to the PEDOT random copolymer embedding in described nano-porous films.

Description

Conductive hydrogel for affine sensing
Cross-Reference to Related Applications
This application claims the priority of the U.S. Provisional Application No. 61/918,099 of December in 2013 submission on the 19th, they are whole Content is totally incorporated herein by reference.
Statement about the right of the lower done invention of federal funding research and development
Inapplicable.
Background of invention
Conducting polymer has the biggest prospect as electrode coating in biomolecular electronics, and provides high conductivity. But, it is characterized in that intrinsic bad mechanical property.Hydrogel may play regulation with the combination of conducting polymer and improve machinery The effect of performance, also provides an antifouling surface and the storage vault of water soluble bioactive agent.In addition, electroactive poly- The blend of compound and biocompatible hydrogel provides has the sensitivity of enhancing and higher biomolecule mobilization Three dimensional structure, is therefore the promising material for developing biosensor.
In all known conducting polymers, poly-(3,4-rthylene dioxythiophene) (PEDOT) shows low-down solid Having cytotoxicity, NIP reaction after implantation, so that they are suitable for bio-sensing and application of biological engineering.Polyethylene Glycol (PEG) be a kind of with excellent antifouling property famous bioavailable polymer.At tissue engineering and biosensor In exploitation, PEG is widely used in minimizing excess protein matter absorption and cell adhesion.It is believed that the group of PEDOT and PEG performance Conjunction can provide promising biological sensing material.
Invention summary
In one embodiment, the invention provides a kind of sensor, this sensor has base plate；At least one contact The electrode of described base plate；Cover the nano-porous films of described electrode；And the bio-identification part selected from lower group (biorecognition element): peptide, antibody, enzyme and fit, wherein said bio-identification some covalent is attached to described Electrode, or it is covalently bound to the PEDOT random copolymer embedding in described nano-porous films, described PEDOT random copolymer has There is a Formulas I structure:
Wherein, each R is independently selected from lower group: OH and described bio-identification part, and at least one of which R is bio-identification Part, and x and y independently be the integer from about 1 to about 1000, and wherein x and y sum is about the integer of 2 to about 1000.
In another embodiment, the present invention provides a kind of for detecting the method for disease marker in biological specimen, including Inventive sensor is contacted with biological specimen, and detects the combination to bio-identification part of the described disease marker, thus examine Survey described disease marker.
In another embodiment, the present invention provides a kind of conductive hydrogel, including the PEG of covalent cross-linking (PEG) hydrogel；With poly-(3,4-ethene dioxythiophene) (PEDOT) random copolymer embedding described PEG-hydrogel；Described PEDOT random copolymer has a Formulas I structure:
Wherein, each R is independently selected from lower group: OH and bio-identification part, and described bio-identification part is selected from lower group: peptide, Antibody, enzyme and fit, at least one of which R is biological identification division, and x and y independently be from about 1 to about 1000 whole Number, wherein x and y sum is about the integer of 2 to about 1000.
In another embodiment, the present invention provides a kind of method preparing conducting polymer of the present invention, including: Suitably form under the polymerizing condition of PEG-hydrogel, PEG-diacrylate is contacted with light trigger；It is embedded into being enough to be formed Under the electropolymerizatioconditions conditions of poly-(3,4-rthylene dioxythiophene) (PEDOT) random copolymer in described PEG-hydrogel, by described PEG-hydrogel with containing 3,4-ethene dioxythiophene (EDOT) and 2,3-dihydro-thiophene also [3,4-b] [1,4] dioxane-2-carboxylic The solution contact of acid (EDOT-COOH), described poly-(3,4-rthylene dioxythiophene) (PEDOT) random copolymer has a Formulas I:
Wherein, each R is OH, and x and y is respectively the integer from about 1 to about 1000, and wherein x and y sum is about 2 to about The integer of 1000；Further, under conditions of being enough to bio-identification some covalent is bound to described PEDOT random copolymer, will Described hydrogel and described bio-identification part contact, thus form the PEDOT random copolymer of Formulas I, at least one of which R is Bio-identification part selected from lower group: peptide, antibody and fit, thus prepare the conductive hydrogel of the present invention.
Accompanying drawing is sketched
Fig. 1 shows assembling and the polymerization of porous polyethylene glycol hydrogel of patterned electrodes.
Fig. 2 A-2C shows that EDOT/EDOT-COOH is to the polymerization in polyethylene glycol hydrogel.
Fig. 3 A and 3B shows cyclic voltammetry curve (sweep speed 50mV/s, the 1X of polyethylene glycol hydrogel electrode PBS), (3A) is to mix without PEDOT, and (3B) mixes for there being PEDOT.
Fig. 4 A and 4B shows the PEDOT-polyethylene glycol hydrogel the securing antibody response to different recombinant concentration.Figure 4A shows along with the increase of restructuring B-IFN-γ concentration, in the cyclic voltammogram of PEDOT-polyethylene glycol hydrogel, and performance Go out to reduce the suppression of peak value.Fig. 4 B shows the reduction current value of the function as restructuring B-IFN-γ concentration.
Fig. 5 shows with the porous plate array of (on-plate) electrode design and parts on plate.(1) bottom-graphical Electrode.These include, working electrode, antielectrode and reference electrode.(2) second layer (blue), nano-porous films, prevents spontaneous Cell in reason liquid is adsorbed onto on electrode.(3) plastic microporous of cut, on the base plate before being adhered to, and (4) mould Material seals, to prevent the physiological fluid evaporation in detection.
Fig. 6 A-6C shows octal plate electrode array structure thing, and it is with engagement pad and connects wire (Fig. 6 A) and silk The reference electrode with Ag/AgCl slurry of wire mark brush, red circle shows that each orifice plate includes working electrode, antielectrode and reference Electrode (Fig. 6 B).Nano-porous films and PDMS microporous membrane parts (Fig. 6 C).
Fig. 7 A and 7B.Fig. 7 A shows that the photopolymerization by PEG and the electropolymerization of the PEDOT in the PEG of preparation are made Standby PEDOT/PEG conductive hydrogel.Fig. 7 B shows, cattle IFN-γ and the combination of the conductive hydrogel securing antibody, causes electricity Chemical signal declines.
Fig. 8 A-8C shows electrode features.Fig. 8 A shows graphical Au electrod-array, and EDOT/PEG conduction water-setting The electrode of (Fig. 8 B) and (Fig. 8 C) afterwards before glue electropolymerization, described conductive hydrogel by the photopolymerization of PEG and PEDOT in system Electropolymerization (in ITO electrode) in standby PEG and prepare (scale: 500 μm).
Fig. 9 A-9D shows SEM image, represents following configuration of surface: gold electrode (Fig. 9 A), PEG (Fig. 9 B), PEDOT (scheme 9C) with PEDOT/PEG (Fig. 9 D) conductive hydrogel.
Figure 10 A-10D shows the sign of conductive hydrogel: (Figure 10 A) cyclic voltammetry curve (CV) show only PEG and The contrast on PEDOT/PEG surface.The cyclic voltammetry curve of PEDOT/PEG substrate shows oxidation intrinsic for PEDOT and reduction, with Only PEG group matter compares current value enhancing 100 times.(Figure 10 B) circulation of PEDOT and PEDOT/PEG of electropolymerization on Au surface Volt-ampere curve.(Figure 10 C) is fixed with the recombinant bovine IFN-of the conducting polymer hydrogel surface variable concentrations of B-INF-gamma antibodies γ excites, and records cyclic voltammetry curve.The capture of IFN-γ causes IpThe decline of value.(Figure 10 D) is based on B-INF-γ Concentration compares IpIt is worth to calibration trace.
Figure 11 A-11E shows the performance test of conductive hydrogel sensor.(Figure 11 A) excites PEDOT/PEG with whole blood Time, electrochemical source of current peak value does not change.(Figure 11 B) the non-specific cell factor or (Figure 11 C) people INF-γ are really in sensing Obvious electrochemical signals is demonstrated on device.Declining in response to the cattle IFN-γ in plasma sample of (Figure 11 D) electrochemical signals. Association between ELISA result and electrochemical signals decline.(Figure 11 E) be the reality of the cattle IFN-γ of cell release from cattle PBMCs Time monitoring.
Figure 12 A-12C shows PEDOT (Figure 12 A), PEG (Figure 12 B) and the AFM mechanics of PEDOT+PEG (Figure 12 C).
Figure 13 A-13D shows the optimization that PEDOT deposits, as the function of the electric charge applied.
Figure 14 A-14B shows by hydroxy-acid group quantitative on the Toluidine blue staining conductive hydrogel that carries out of test.
Figure 15 A-15B shows the fluoroscopic image under the cattle INF-γ of variable concentrations in ITO electrode.
Figure 16 A-16B shows the INF-γ detection being mixed with Sanguis Bovis seu Bubali/PBS (1:1).
Figure 17 shows the schematic diagram of inventive sensor, including the position of reference electrode, working electrode and antielectrode.
Figure 18 A-18B shows the different embodiments (cross section) of biosensor of the present invention (100), including base plate (110), electrode (120), nano-porous films (130) with bio-identification part (140), and the self assembly being positioned on base plate Monolayer (monolayer) (150).
Figure 19 A-19B shows the different embodiments (cross section) of the biosensor (200) of the present invention, including base plate (210), electrode (220), nano-porous films (230), wherein bio-identification part (240) is by the self-composed monomolecular on electrode Layer (250) is connected to electrode.
Detailed Description Of The Invention
I. summarize
The present invention provides a kind of for biological substance detection and the sensor of disease detection.Described sensor includes biological knowledge Other part, this bio-identification part is attached to cover the conducting polymer of electrode, thus this bio-identification part and biological substance Combination cause the electrode potential detected to change, combining movement (binding event) therefore detected.This sensor Label can not be used to detect.Additionally, conducting polymer, PEDOT-polyethylene glycol hydrogel, there is the mechanics of improvement Performance, and owing to being covalently bond to backplate surface, it can be better against the leafing with electrode.
II. define
" base plate " refers to any solid material being suitable to support inventive sensor.Suitable base plate can include such as The materials such as silicon, silicon dioxide and indium tin oxide.The example of suitable base plate includes but not limited to, glass (includes porosity-controllable Glass), polymer (such as, polystyrene, polyurethane, polystyrene-divinylbenzene copolymer), silicone rubber, Quartz, latex, transition metal, magnetic material, silicon dioxide, silicon nitride, GaAs and derivant thereof.Except the activity on surface Outside position, baseboard material generally has toleration to the various chemical reaction conditions that can suffer from.The end used in the present invention Plate can be smooth or coarse.Smooth surface is to have on this surface few to cause coarse feature.Rough surface is There is on this surface the substantial amounts of feature producing friction.The base plate of the present invention includes the first base plate and the second base plate.Described base plate Can be flat or injustice, flexible or rigidity.Additionally, described base plate can be porous, netted or textile-like. Described base plate can be opaque or transparent, and can be with transmission or reflection light, or both have concurrently.Those skilled in the art will Understand and the present invention can use other base plates.
" electrode " refers to the electric conductor for contacting with a part for circuit.Described circuit generally can include some one The electrode worked, such as working electrode, antielectrode and reference electrode.Working electrode is reaction interested in system or phase interaction With the electrode occurred.Antielectrode assists working electrode in the measurement carried out.Reference electrode provides stable electrode potential, phase Working electrode and the electromotive force of antielectrode are measured for this electrode potential.
" film " refers to layer, and this layer allows material and fluid to move to another location from a position by film.Due to hole Size limitation, described film can stop the movement of some materials.When described hole is nanosized, described film can be that nanometer is many Pore membrane.
" contact " and refer to allow at least two difference object in close proximity to so that the one of at least one surface of the first object Part contacts with the part at least one surface of the second object.
" covalent bond " refers to the formation of covalent bond between base plate of the present invention and a part of hydrogel.
" bio-identification part " refer to can specific bond to the biological substance of another kind of biological substance.
" polypeptide ", " peptide " and " albumen ", used interchangeably herein, refers to the polymer of amino acid residue.All this three Individual term is applicable to amino acid polymer, and wherein one or more amino acid residues are corresponding naturally occurring amino acid whose people Work chemical simulation thing, and naturally occurring amino acid polymer and the amino acid polymer of non-naturally-occurring.It is as used herein, This term covers the amino acid chain of random length, and including full-length proteins, wherein amino acid residue is connected by covalent peptide bonds.
As used herein, " antibody " includes having immunoreactive immunoglobulin molecules, including many with specific antigen Clonal antibody and monoclonal antibody.This term also includes genetically engineered form, such as chimeric antibody (such as, humanized murine Body) and conjugation of antibodies (such as, bi-specific antibody).This term " antibody " also includes the antigen binding forms of antibody, including tool There is the fragment (such as, Fab ', F (ab ') of antigen binding capacity2, Fab, Fv and rIgG.Separately refer to, Pierce Catalog And Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL). separately refer to, such as, Kuby, J., Immunology, 3rdEd., W.H.Freeman&Co, New York (1998).This term also refers to recombinant single chain Fv fragment (scFv).Term antibody also includes bivalence or bispecific molecule, double antibody, three antibody and four antibody.Bivalence and bispecific Molecule is described in J Immunol 148:1547, Pack and Pluckthun (1992) biochemistrys 31 such as such as Kostelny: 1579, Hollinger etc., 1993, supra, Gruber etc. (1994) J Immunol:5368, Zhu etc. (1997) Protein Sci 6:781, Hu etc. (1996) Cancer Res.56:3055, Adams etc. (1993) Cancer Res.53:4026, and McCartney, etc. (1995) Protein Eng.8:301.
With specific antigen, there is immunoreactive antibody to be produced by recombination method, such as in phage or similar load The recombinant antibodies storehouse of body selects, see, e.g. Huse etc., Science 246:1275-1281 (1989)；Ward etc., Nature 341:544-546(1989)；With V gold ghan etc., Nature Biotech.14:309-314 (1996), or by use The DNA immunization animal of antigen or coding for antigens.
Typically, immunoglobulin has heavy chain and light chain.Every heavy chain and light chain contain constant region and variable region (these Region is also referred to as " domain ").Light chain and variable region of heavy chain include by three hypervariable regions (also referred to as " complementary determining region " or " CDRs ") four " framework " districts interrupting.Defined framework region and the scope of CDRs.Different light chains or the framework of heavy chain in species Region sequence is guarded relatively.The framework region of antibody, i.e. as light chain and the group frame district of heavy chain of component, in three-dimensional Position in space and align CDRs.
CDRs is mainly responsible for being bound to epitope.CDRs so-called CDR1, CDR2 and CDR3 of every chain, order is compiled Number from the beginning of N-terminal, and the chain being positioned at typically also through specific CDR identifies.Therefore, VHCDR3 is positioned at it and is sent out The variable domains of the existing heavy chain in antibody, and VLCDR1 comes from the variable knot of the light chain in its antibody being found The CDR1 in structure territory.
Mention " VH" or " VH " refer to the variable region of heavy chain immunoglobulin of antibody, including Fv, scFv or Fab Heavy chain.Mention " VL" or " VL " refer to the variable region of light chain immunoglobulin, including the light chain of Fv, scFv, dsFv or Fab.
Phrase " scFv " or " scFv " refer to antibody, the heavy chain of two the most traditional chain antibodies and light chain variable District has combined one chain of formation.Typically, joint peptide is inserted between two chains, to allow suitably fold and enliven binding site Generation.
" chimeric antibody " is immunoglobulin molecules, wherein (a) constant region or one part, is changed, substitutes or exchanges, Thus antigen binding site (variable region) is connected to the constant of different or that change classification, effector function and/or species District, or connect most chimeric antibody and impart the diverse molecule of new features, such as, enzyme, toxin, hormone, growth because of Son, medicine etc.；Or (b) variable region or one part, changes with the variable region with antigenic specificity that is different or that change Become, substitute or exchange.
" humanized antibody " is immunoglobulin molecules, and it includes the minmal sequence from non-human immunoglobulin.People Source antibody includes human normal immunoglobulin's (receptor antibody), and the residue of the complementary determining region (CDR) wherein carrying out autoreceptor is had Non-human species's (donor antibody) (such as mice, rat or rabbit) the CDR residue of desired specificity, affinity and ability is taken Generation.In some instances, the Fv Framework residues of human normal immunoglobulin is replaced by corresponding non-human residues.Humanized antibody also may be used Comprise the residue neither not found in the CDR introduced or Frame sequence at receptor antibody.Generally, humanized antibody can comprise At least one, essentially all (region) of typically two variable regions, the most all or essentially all of CDR region territory Being equivalent to the region of non-human immunoglobulin, all or essentially all of framework (FR) region is that human normal immunoglobulin has Those of sequence.Most preferably, humanized antibody also includes at least some of of constant region for immunoglobulin (Fc), is typically people (Jones etc., the Nature 321:522-525 (1986) of immunoglobulin；Riechmann etc., Nature 332:323-329 (1988)；And Presta, Curr.Op.Struct.Biol.2:593-596 (1992)).Humanization can substantially follow Winter Method (Jones etc., the Nature321:522-525 (1986) worked together with it；Riechmann etc., Nature 332:323-327 (1988)；Verhoeyen etc., Science 239:1534-1536 (1988)), by by rodentine CDRs or CDR sequence Replace the corresponding sequence of people's antibody.Therefore, this kind of humanized antibody is chimeric antibody (U.S. Patent number 4,816,567), wherein It is substantially less than whole person variable region (region) (substantially less than an intact human Variable domain) replaced by the corresponding sequence from non-human species.
" epi-position " or " antigenic determinant " refers to the site on antigen, and antibodies is in this site.Epi-position can be by even Continuous aminoacid or the non-contiguous amino acids arranged side by side formation by the three of albumen grades of foldings.The epi-position formed by continuous amino acid exists Usually keep when being exposed to denaturing solvent, and folded the epi-position formed by three grades and would generally lose under denaturing solvent processes Lose.Epi-position generally includes at least 3 at unique space conformation, more generally, and at least 5 or 8-10 aminoacid.Measure The method of epi-position space conformation includes: such as, X-ray crystallography and two dimensional NMR.See, e.g., table in molecular biology Position drawing method (Epitope Mapping Protocols in Methods in Molecular Biology), rolls up .66, Glenn E.Morris,Ed(1996)。
" enzyme " refers to biocatalyzer.
" fit " refers to be bound to oligonucleotide or the peptide of certain target molecules.
" 3,4-ethene dioxythiophene (EDOT) " refers to following chemical constitution:
" 2,3-dihydro-thiophene also [3,4-b] [1,4] dioxane-2-carboxylic acid (EDOT-COOH) " refers to following chemistry knot Structure:
" PEDOT random copolymer " refers to poly-(3,4-rthylene dioxythiophene) (PEDOT) copolymer, wherein EDOT and EDOT-COOH copolymerization forms PEDOT, described PEDOT together and has an EDOT-COOH of random distribution in the copolymer:
" self assembled monolayer " refers to the material such as organic compound of monolayer, is assembled on backplate surface.From group Dress monolayer can be formed from various materials, the such as silane on silicon or the mercaptan on gold.
" oxidoreduction report section (Redox reporting moiety) " referring to can be in response to the change of electromotive force And produce the arbitrary portion that can measure signal.Measurable change of electric current can be associated with the concentration of target analytes.
" thiol moiety " refers to "-SH " group.
" PEG " refers to Polyethylene Glycol and polyethylene oxide polymer.PEG can have from less than 1000 dalton to being more than 100,000 the most suitable daltonian molecular weight.PEG also can be functionalized at α end or ω end with various groups.Such as, PEG can be by one or more acrylate group functionalizations.When by two acrylate group functionalizations PEG, form poly-second Glycol-diacrylate.PEG can be mixed the polymeric matrix of crosslinking by the crosslinking of PEG chain and water and be formed hydrogel.
As used herein, term " hydrogel " refers to the most complementary and interconnective two-phase substrate, described Substrate has existing hydrophilic and has again hydrophobic solid constituent (usually polymer, the most highly cross-linked polymerization Thing).Additionally, described substrate contains the liquid component (such as water) being retained in substrate by molecular separating force.Hydrophobic property is base Matter provides a certain degree of water-insoluble, and hydrophilic nmature provides water penetration.It will be understood by those skilled in the art that several not Can be combined with the polymer of type and use to form hydrogel available in the inventive method.
" microtitration orifice plate " refers to the plate with multiple holes for test purposes, and wherein electrode is at the end in each hole Portion.Each electrode can be surrounded by border, hole, and described hole borders is around the wall of electrode.Each hole can be covered by port lid.
" disease marker " refers to the existence of specified disease or measurable index of relative risk.Disease marker can be Heredity, or represented relative to the relative concentration of normal concentration by certain component in patient blood or health.Described disease Labelling can also be certain organs or the relative function (relative to this organ or the normal function of process) of process in patient body.
" biological specimen " refers to the arbitrary sample of patient body, including tissue, organ, liquid etc..
" peak reduction current " refers to the electric current on the formal potential of electrochemical substance that is just being reduced.
" square wave voltammetry " refers to the waveform used in electrochemistry, and electromotive force (voltage) is the most in a linear fashion and with zigzag Mode scan.This potential scan method makes capacitive current minimize, and the sensitive measurement for electric current minor variations is excellent Choosing.
" tuberculosis " refers to be drawn by mycobacteria such as mycobacterium tuberculosis (myobacterium tuberculosis) The disease risen.
" hepatitis C " refers to the disease caused by hepatitis C virus (member of flaviviridae (Flaviviridae)) Sick.
" IFN-γ " and " IFN-γ " refers to combine the II type interferon of II type IFN receptor, interference host cell Virus infects.IFN-γ regulates multiple biological function, such as antiviral response, cell growth, immunne response and tumor suppression, and Multiple human diseases can be mediated.
" light trigger " starts the compound of polymerization process after referring to irradiate.Light trigger can produce acid, and (light-acid produces Agent or PAG) or free radical, and other releasers.Described acid, free radical or other releasers, cause polymerization to make afterwards With.
The polymerization process that " electropolymerizatioconditions conditions " refers to electrically activate and non-chemically start.The condition of electropolymerization includes with fully Electric current and/or electric charge application electromotive force to pre-polymeric mixture to cause polymerization.Electropolymerizatioconditions conditions can become with the polymer of preparation Change.
" embed (embedded) " and refer to conducting polymer intercalation (intercalation) in nano-porous films, Wherein said conducting polymer is not covalently attached to nano-porous films.
III. conductive biological sensor
The present invention provides a kind of biosensor, may indicate that the biological substance of specified disease for detection.
In some embodiments, the present invention provides a kind of sensor, and this sensor has base plate；At least one contact institute State the electrode of base plate；Cover the nano-porous films of described electrode；And selected from the bio-identification part of lower group: peptide, antibody, enzyme, With fit, wherein said bio-identification some covalent is attached on described electrode, or is covalently bound to embed described nanoporous PEDOT random copolymer in film, described PEDOT random copolymer has a Formulas I structure:
Wherein, each R is independently selected from lower group: OH and described bio-identification part, and at least one of which R is bio-identification Part, and x and y independently be the integer from about 1 to about 1000, and wherein x and y sum is about the integer of 2 to about 1000.
Figure 18 A-18B shows the different embodiments (cross section) of the biosensor (100) of the present invention, including base plate (110), electrode (120), nano-porous films (130) with bio-identification part (140), and the self assembly being positioned on base plate Monolayer (150).Figure 19 A-19B also show the different embodiments (cross section) of the biosensor (200) of the present invention, Including base plate (210), electrode (220), nano-porous films (230), wherein bio-identification part (240) by electrode from group Dress monolayer (250) is connected to electrode.In some embodiments, described bio-identification part (240) may be connected directly to Electrode (220), it is not necessary to the self assembled monolayer (seeing Figure 19 B) of intervention.
The base plate of the present invention can be adapted for supporting electrode and combining any base plate of polyethylene glycol hydrogel.The suitable end The example of plate can be glass, insulator, pottery, quasiconductor, metal, polymer etc..Representational base plate includes glass.One In a little embodiments, described base plate can be glass.
The biosensor of the present invention can include the electrode (120) of the contact base plate of any proper number and type.Example As, described biosensor can include working electrode, antielectrode and reference electrode.Described electrode can be by any suitable conduction material Material is made.Such as, described electrode can be made of metal, such as but not limited to gold, silver, platinum, copper etc..Other electrode material includes Slaine, such as silver chloride.In some embodiments, described biosensor includes working electrode, antielectrode and reference electricity Pole.In some embodiments, described electrode is made of gold.
The electrode of the present invention can orient the most in any suitable form.Such as, described electrode can be round Shape, by another electrode retaining collar around an electrode.Additionally, described electrode can be any suitable shape, as square, circular, Or toroid.
In some embodiments, described electrode is the gold electrode of pattern.In some embodiments, described sensing Device includes central authorities' working electrode, the antielectrode of cincture and reference electrode.In some embodiments, described working electrode is gold Electrode.In some embodiments, described reference electrode is silver/silver chloride electrode.
The biosensor of the present invention may also comprise the nano-porous films covering electrode.Described nano-porous films can be from any Prepared by suitable material.Such as, nano-porous films can be polymer, inorganic material (such as pottery) or oxide is (such as Aluminium oxide) or other similar materials.Described nano-porous films can be conduction or nonconducting.Described nano-porous films can There is the hole of any suitable dimension, as from 10nm to 100 μm.Described nano-porous films can be any suitable thickness, such as From 10nm to 10mm.In some embodiments, described nano-porous films can be aluminium oxide.In some embodiments, originally The biosensor of invention does not include nano-porous films.
Described nano-porous films can also be conductive hydrogel.Any suitable conductive hydrogel can be used in the present invention. Described hydrogel can be prepared by any suitable material, such as Polyethylene Glycol (PEG).Described PEG can by crosslinkable groups (such as Acrylate, methacrylate or other polymerizable groups) functionalization.Described PEG can be any suitable molecular weight.Example As, described PEG can have the molecular weight of about 1kDs, 2,3,4,5,6,7,8,9 or 10kDa.Described PEG also can have at most about The molecular weight of 100kDa.In some embodiments, described PEG can be the Polyethylene Glycol-dipropyl with about 6kDa molecular weight Olefin(e) acid ester.
In some embodiments, polyethylene glycol hydrogel can be covalently attached to base plate.Such as, described base plate can be with from group Dress monolayer is modified, and described self assembled monolayer can be covalently bond in the polymerization process of preparation PEG-hydrogel PEG-diacrylate.In some embodiments, the described base plate self assembled monolayer containing acrylate-functional groups is repaiied Decorations.Nano-porous films is covalently attached to base plate can reduce the leafing of nano-porous films, improves stablizing of biosensor of the present invention Property and life-span.
Conductive hydrogel may also include conducting polymer.Any suitable conducting polymer substrate is all applicable to the present invention's Biosensor.Such as, conducting polymer can include poly-(3,4-rthylene dioxythiophene) (PEDOT) polymer.The present invention is also Other conducting polymer can be used, include but not limited to, polypyrrole, polythiophene etc..
Described conductive hydrogel can by any suitable by the way of prepare.For example, it is possible to first by vinyl polymerization side Method prepares polyethylene glycol hydrogel, uses electropolymerization method to prepare PEDOT polymer subsequently.Ethene polymerization method includes acid catalysis Or free radical cause polymerization.In some embodiments, the polymerization of PEG can be light-initiated.PEDOT polymer is permissible It it is the copolymer of several EDOT monomeric unit (including EDOT and EDOT-COOH).When EDOT monomer copolymerization, it is possible to formed PEDOT random copolymer.PEDOT copolymer can be any suitable molecular weight, have any suitable distribution EDOT and EDOT-COOH comonomer.Such as, the ratio of EDOT and EDOT-COOH comonomer can from about 1000:1 to about 1:1000, Or about 500:1,100:1,50:1,10:1,9:1,8:1,7:1,6:1,5:1,4:1,3:1,2:1,1:1,1:2,1:3,1:4, 1:5,1:6,1:7,1:8,1:9,1:10,1:50,1:100 or about 1:1000.It is used as the monomer of other ratios.
In some embodiments, PEDOT random copolymer can have a Formulas I structure:
Wherein, each R is independently selected from lower group: OH and bio-identification part, and at least one of which R is biological identification division, Further, x and y independently be the integer from about 1 to about 1000, and wherein x and y sum is about the integer of 2 to about 1000.Real at some Executing in mode, the ratio of x Yu y is from about 10:1 to about 1:10.In some embodiments, the ratio of x Yu y is about 1:4.
PEDOT random copolymer can be prepared before or after prepared by polyethylene glycol hydrogel.In some embodiments, PEDOT random copolymer is prepared after prepared by polyethylene glycol hydrogel, by being gathered by EDOT and EDOT-COOH monomer deposition Electropolymerization conduction (conductions) it is exposed in ethylene glycol hydrogel and by polyethylene glycol hydrogel, thus PEDOT is random Copolymer is prepared in polyethylene glycol hydrogel, but is not covalently attached to polyethylene glycol hydrogel.
The present invention includes any suitable bio-identification part.Such as, described bio-identification part can include antibody, fit Body, camellid antibody (camelid), peptide, albumen or other biological substance.In some embodiments, described biological knowledge Part can not be peptide, antibody, enzyme or fit.In some embodiments, described bio-identification part can be antibody.One In a little embodiments, described bio-identification part can be enzyme.In some embodiments, described bio-identification part can be Fit.In some embodiments, described bio-identification part can be IFN-γ.In some embodiments, described biology Identification division can be cattle-IFN-γ.
The present invention can use any suitable fit or enzyme as bio-identification part.Fit in the present invention can be to inflammation Disease cytokine has specificity, described inflammatory cytokine include but not limited to TNF-α, IL-2, IL-4, IL-6, IL-10, IL-17, IL-21 and TGF-β.Described fit also can have specificity, described cell surface marker to cell surface marker Include but not limited to CD4, CD8, CD36, CD 14, CD45 and EpCAM.Described fit also pathogen component can be had special Property, described pathogen component includes but not limited to HIV, HBV, HCV and sexually transmitted disease (STD) (STDs).Fit also can be thin to being present in The transmembrane protein such as CD63 of the outer vesicle of born of the same parents has specificity.Any oxidoreductase can be used as the bio-identification portion in the present invention Point.The primary categories of described enzyme includes but not limited to: oxidoreductase (such as peroxidase, glucoseoxidase, lactic acid oxygen Change enzyme) and NADH dependent enzyme.
Other biological identification division includes peptide.Described peptide can be to be bound to disease marker, or as protease substrate Any suitable peptide.This type of peptide allows the biosensor detection proteinase activity of the present invention.
Described bio-identification part may be coupled to conducting polymer, or is directly connected to electrode.At some embodiments In, bio-identification part may be coupled to conducting polymer.When described conducting polymer is PEDOT random copolymer, described Bio-identification part may be connected to the hydroxy-acid group of PEDOT random copolymer.In some embodiments, described bio-identification portion Dividing the PEDOT random copolymer being covalently bound to be embedded in nano-porous films, wherein said nano-porous films includes having The polyethylene glycol hydrogel of the PEG chain of about 1000Da to about 10,000Da molecular weight.
In some embodiments, described bio-identification part can be covalently attached on electrode.When described bio-identification portion When point being covalently attached to electrode, described electrode can be repaiied by the self assembled monolayer that can be covalently attached to bio-identification part Decorations.In some embodiments, described bio-identification some covalent is connected on electrode form bio-identification portion on electrode The self assembled monolayer divided.In some embodiments, described bio-identification part for have oxidoreduction report section and Thiol moiety fit, wherein said thiol moiety is covalently attached to metal working electrode.
The biosensor of the present invention can have a bio-identification part or several different bio-identification part.Example As, PEDOT conducting polymer can be by several different bio-identification part functionalizations.This allows to detect several different disease simultaneously Sick labelling, and have greater flexibility for the moment when detecting several different disease markers.Similarly, when described bio-identification When part is joined directly on electrode, each electrode can be repaiied by a kind of bio-identification part or several different bio-identification part Decorations.See, e.g. Liu etc., " from the electrode of identical fit-functionalization, detecting the cytokine profiles of emiocytosis " Biosensors and Bioelectronics 2015,64,43-50, includes in herein in the way of entirety is quoted.
The biosensor of the present invention can include other components multiple.Such as, described biosensor can include that trace drips Determine orifice plate, port lid, etc..In some embodiments, described biosensor also includes for accommodating the micro-of biosensor Amount titration orifice plate.In some embodiments, described biosensor also includes that port lid, described port lid comprise one or more connecing Bottoming out the wall of plate, described wall defines the border, hole around electrode.
IV. the method detecting disease marker
Present invention also offers the method using biosensor of the present invention to detect disease by detection disease marker.? In some embodiments, the present invention provides a kind of method of disease marker detected in biological specimen, senses including by the present invention Device contacts with biological specimen, and detects the combination of described disease marker and bio-identification part, thus detects described disease mark Note.
Described detection can be carried out by any suitable device or equipment.Such as, described detection can include detecting by this The change of the signal of telecommunication that invention biosensor produces.Without being bound by theory, when described disease marker is attached to bio-identification During part, the signal of telecommunication in nano-porous films produces change, and this change is passed from bio-identification part by conducting polymer Deliver to electrode.
In some embodiments, detection disease marker includes measuring PEDOT randomly altogether with the combination of bio-identification part The peak value reduction current of polymers.In some embodiments, square wave voltammetry is utilized to detect described disease marker and bio-identification The combination of part.
Any suitable disease marker can be used in detection.Such as, described disease marker can be from the peptide of patient, egg In vain, antibody, enzyme, cell, tissue etc..In some embodiments, described disease marker can be interferon.Some embodiment party In formula, described disease marker can be IFN-γ.Other diseases labelling includes ectosome (exosomes), extracellular vesicle (EVs).Described extracellular vesicle comprises the specific fit transmembrane protein that can detect that of use.Exemplary transmembrane protein include but It is not limited to CD63.Other diseases labelling includes protease, such as but not limited to, matrix metalloproteinase (MMPs) MMP2, MMP4 And MMP9, and urokinase (uPA).
Described disease marker may indicate that the existence of any disease, and such as cancer, virus infect, antibacterial infects, heart disease Disease, autoimmune disease etc..Representational virus infects and includes but not limited to: HIV, HBV, HCV, herpes, chickenpox banding bleb Exanthema virus, Chinese mugwort Pasteur virus (Epstein-barr), cytomegalovirus, human papillomavirus, BK virus, JC virus, smallpox virus, Coxsackie virus, poliomyelitis, rhinoceros virus, SARS, yellow fever, dengue fever, West Nile, rubella, influenza, angstrom Bo La, Marburg, measles, mumps, respiratory syncytial virus, metapneumovirus, rabies and other.Representative Antibacterial infect include but not limited to, pulmonary tuberculosis, tetanus, typhoid fever, diphtheria, syphilis and leprosy.Representative cancer include but It is not limited to, leukemia, CNS, kidney, nonsmall-cell lung cancer, melanoma, carcinoma of prostate, renal carcinoma, hepatocarcinoma, breast carcinoma, ovary Cancer, cancer of pancreas or intestinal cancer.Other diseases
In some embodiments, described disease marker shows by pulmonary tuberculosis or hepatitis C infections.In some embodiments, Described disease marker includes IFN-γ.In some embodiments, described bio-identification part includes interferon-γ (IFN-γ) Antibody.In some embodiments, described bio-identification part includes being specific to the fit of IFN-γ.
V. conductive hydrogel
The present invention also provides for the conductive hydrogel for biosensor of the present invention.In some embodiments, the present invention There is provided a kind of conductive hydrogel, including PEG (PEG) hydrogel of covalent cross-linking；With the described PEG-hydrogel of embedding Poly-(3,4-rthylene dioxythiophene) (PEDOT) random copolymer, described PEDOT random copolymer has a Formulas I structure:
Wherein, each R is independently selected from lower group: OH and bio-identification part, and described bio-identification part is selected from lower group: peptide, Antibody, enzyme and fit, at least one of which R is biological identification division, and, it is whole that x and y independently be from about 1 to about 1000 Number, wherein x and y sum is about the integer of 2 to about 1000.
In some embodiments, described polyethylene glycol hydrogel includes having from about 1000Da to about 10,000Da molecule The PEG chain of amount.In some embodiments, described polyethylene glycol hydrogel includes the PEG chain with about 6000Da molecular weight.? In some embodiments, the ratio of x Yu y is from about 10:1 to about 1:10.In some embodiments, the ratio of x Yu y is about 1:4. In some embodiments, the PEG hydrogel of described covalent cross-linking is prepared by Polyethylene Glycol-diacrylate.
VI. the method preparing conductive hydrogel biosensor
The conductive hydrogel of the present invention can be prepared by any method well known by persons skilled in the art.For example, it is possible to First prepare polyethylene glycol hydrogel, then conducting polymer, such as PEDOT, can be polymerized in polyethylene glycol hydrogel, Thus conducting polymer embeds in polyethylene glycol hydrogel but is not covalently attached to polyethylene glycol hydrogel.
In some embodiments, the invention provides a kind of method preparing conductive hydrogel of the present invention, including: suitable Under the polymerizing condition forming polyethylene glycol hydrogel, Polyethylene Glycol-diacrylate is contacted with light trigger；Be enough to shape Become the electropolymerizatioconditions conditions of poly-(3,4-ethene dioxythiophene) (PEDOT) random copolymer being embedded in polyethylene glycol hydrogel Under, by polyethylene glycol hydrogel and containing 3,4-rthylene dioxythiophene (EDOT) and 2,3-dihydro-thiophene also [3,4-b] [Isosorbide-5-Nitrae] two The solution contact of alkane-2-carboxylic acid (EDOT-COOH), described poly-(3,4-rthylene dioxythiophene) (PEDOT) random copolymer has There is a Formulas I:
Wherein, each R is OH, and, x and y is respectively integer from about 1 to about 1000, and wherein x and y sum is about 2 and arrives The integer of about 1000；And under conditions of being enough to bio-identification some covalent is bound to PEDOT random copolymer, by water-setting Glue and bio-identification part contact, thus form the PEDOT random copolymer of Formulas I, at least one of which R is the life selected from lower group Thing identification division: peptide, antibody and fit, thus prepare the conductive hydrogel of the present invention.
Described Polyethylene Glycol-diacrylate can be any suitable Polyethylene Glycol-diacrylate with following formula:
The polyalkylene glycol moiety of described Polyethylene Glycol-diacrylate can have the most suitable molecule as above Amount.In some embodiments, described Polyethylene Glycol-diacrylate has from about 1000Da to about 10, the molecule of 000Da Amount.In some embodiments, described Polyethylene Glycol-diacrylate has the molecular weight of about 6000Da.
Described polyethylene glycol hydrogel can be prepared by any method well known by persons skilled in the art.Such as, use Suitable polymerizable groups can at one end or two ends functionalization PEG.Suitable polymerizable groups includes but not limited to, acrylic acid Ester, methacrylate, styrene, cyanoacrylate and other.The polymerization of polyethylene glycol hydrogel can radical polymerization, Acid or alkali polymerizing condition under carry out.In some embodiments, described polyreaction is carried out under the conditions of radical polymerization.? In some embodiments, the polymerizing condition of described formation polyethylene glycol hydrogel includes irradiating Polyethylene Glycol-diacrylate.
When with light trigger irradiate Polyethylene Glycol-diacrylate prepare polyethylene glycol hydrogel time, described irradiation can Carry out under various conditions.Such as, described polyethylene glycol hydrogel can be irradiated from 1 second to more than 1 minute, including 2,3,4,5, 10,15,20,30,40 and 50 seconds.The present invention also contemplates for irradiating the longer time.The wavelength irradiated and intensity depend on use Light trigger, photoinitiator concentration and polyethylene glycol hydrogel are expected the degree of crosslinking.
Described light trigger can be any suitable light trigger known in the art.Such as, described light trigger can To be azodiisobutyronitrile, benzoyl hydrogen peroxide, camphorquinone or 1-[4-(2-hydroxy ethoxy)-phenyl]-2-hydroxyl-2-first Base-1-propyl group-1-ketone (2959, from vapour bar (Ciba)).
Described conducting polymer can be polymerized by any method well known by persons skilled in the art, such as electropolymerization.As On discussed, described conducting polymer can be single polymer or copolymer.Representational polymer includes polythiophene, poly-pyrrole Cough up and polyhenylene (polyphenylenes).When described conducting polymer is polythiophene based polyalcohol, it is possible to use Ren Heshi When thienyl monomer.Representational thiophene monomer includes but not limited to, thiophene, 3 methyl thiophene, 3,4-rthylene dioxythiophene And 2,3-dihydro-thiophene also [3,4-b] [1,4] dioxane-2-carboxylic acid (EDOT-COOH) (EDOT).Described EDOT-COOH monomer Carboxylic acid functional including available bio-identification part functionalization.
In some embodiments, described conducting polymer is polythiophene polymers.In some embodiments, lead described in Electric polymer is PEDOT copolymer.When described conducting polymer is PEDOT copolymer, described EDOT and EDOT-COOH monomer Ratio as above that can be the most suitable exists.In some embodiments, described EDOT and EDOT-COOH is with from about The ratio of 10:1 to about 1:10 exists.In some embodiments, described EDOT and EDOT-COOH exists with the ratio of about 1:4.
Conducting polymer can use any method bio-identification part well known by persons skilled in the art to modify.Example As, when described conducting polymer is the PEDOT copolymer using EDOT-COOH, described bio-identification part can use amide Form chemistry (amide formation chemistry) and be connected to PEDOT copolymer.The preparation of active ester, such as, pass through EDC Chemistry is known and can be used in the present invention.
Described bio-identification part can be any suitable material described above.In some embodiments, described life Thing identification division is antibody.In some embodiments, described bio-identification part is fit.
VII. embodiment
Embodiment 1: preparation PEDOT-polyethylene glycol hydrogel
In order to develop immunosensor, we construct the compound-type conducting hydrogel of a kind of novelty, and it includes conduction Carboxy-functionalized PEDOT and high molecular weight PEGs.Carboxy-functionalized offer Bioconjugation ability.By at miniaturization gold electrode Top polymerization PEG, uses water-based microemulsion that prepared by PEDOT copolymer electropolymerization biosphere in porous PEG gel subsequently Face.Described PEDOT/PEG copolymer is component adjustable (compositionally tunable), and it is at electrode surface Deposition on array is controlled.The COOH group being positioned on conducting polymer chain passes through active ester group B-IFN-gamma antibodies Adjust (tailored).It is fixed with the conductive hydrogel electrode of antibody for the B-IFN-γ in buffer and Ox blood plasma is carried out Unmarked Electrochemical Detection.B-IFN-γ causes electrochemical signals to reduce to the combination of antibody due to electron transmission obstacle.This The method for sensing planting shutdown signal (signal-off) need not the oxidoreduction labelling of any outside for detecting.
The process manufacturing sensing surface starts from electrode tip photopolymerization polyethylene glycol hydrogel.Subsequently, PEDOT copolymerization Thing is electropolymerization in polyethylene glycol hydrogel.Due to its inherent character, deposition have the electrode of conductive hydrogel when oxidized in Reveal faint blue light, and when reducing, be changed into navy blue (Fig. 7 A).Use scanning electron microscopy (SEM) research conduction poly- Compound and the morphological feature on conductive hydrogel surface.As from SEM image finding (Fig. 7 B), PEDOT deposition on gold electrode Showing coarse granule form, the polyethylene glycol hydrogel of polymerizable ultraviolet shows porous form.PEDOT voltolisation in PEG Close the hole being filled with in PEG.In gold surface, single PEDOT polyreaction is affected by bad mechanical property.
It is essential that the PEDOT molecule in gel occurs redox reaction when applying voltage, and have distinctive Oxidoreduction peak.The cyclic voltammetry curve of the polyethylene glycol hydrogel being not incorporated into and being mixed with electric conductivity PEDOT often demonstrates ratio The conductive hydrogel electrical property (Figure 10 A) that rule polyethylene glycol hydrogel strengthens.In PEG, the incorporation of conducting polymer shows more Strong redox current value, strengthens nearly 100 times.Intrinsic redox characteristic strong for PEDOT is further used for biological biography Sensing is used.PEDOT/ polyethylene glycol hydrogel shows similar CV characteristic with PEDOT, this demonstrate that PEG does not affect PEDOT Electrochemical behavior (Figure 10 B).
The electric charge applied by changing electric polymerization reaction optimizes PEDOT deposition in PEG gel.As from as deposition The data of the Redox behavior of the function of electric charge it can be seen that we can confirm that, electric charge 8 × 10-4Redox property during C Strengthen, and relatively low and higher electric charge (or thickness of PEDOT film) causes oxidoreduction peak and reduction current (Figure 13 A-of difference 13D)。
Prepolymer polyethylene glycol hydrogel solution, 5% Polyethylene Glycol-diacrylate and 1% light trigger in PBS (Irgacure 2959) is coated on the gold electrode of graphical slide.These slides are sudden and violent by the location photomask of top of electrodes It is exposed to ultraviolet radiation (60mJ/cm2) 5 seconds.Then polyethylene glycol hydrogel in the preparation at graphical gold electrode top exists The EDOT-COOH aqueous solution of 10mM is (containing 2.5mM EDOT, 0.05MSDS and 0.1M LiClO4For electric polymerization reaction in) Mode in batches hatch.Use CHI 6044d electrochemical analyser (CH Instruments, Inc. Austin, De Kesa This), under constant 1.1V voltage, in current measurement mode (amperometrically) by this monomer solution electropolymerization to many In the polyethylene glycol hydrogel of hole.Resulting polymers is applying 8 × 10-4There is when C electric charge is to polymeric film the oxidoreduction of enhancing Performance.
Antibody is covalently bound by carboxylic group present on PEDOT.Activated carboxyl is with covalency sessile antibody.Conduction is poly- Compound hydrogel array 0.2mM EDC in deionized water and 0.05mM NHS hatches 15 minutes.Described array PBS floats Wash, and at 4 DEG C by 50 μ g/mL cattle INF-gamma antibodies overnight incubation in PBS.Use CHI instrument at conducting polymer hydrogel Electrochemical Detection is carried out on array.Cyclic voltammetry is used to detect the combination of cattle INF-γ in the range of-0.7V and 0.4V.Often Secondary measurement measures peak point current (I at-0.35Vp), signal is done figure as the function of concentration relative current value.
In order to fix B-IFN-gamma antibodies molecule, the COOH group standard EDC-NHSization being present on conducting polymer Learn and activate.At 1.1V voltage, keep constant charge 8 × 10-4C, makes EDOT-COOH and EDOT containing sodium lauryl sulphate (SDS) LiClO4Mixture in aqueous solution is aggregated in PEG-hydrogel layer in current measurement mode.The method has obtained Interconnective polymer architecture in polyethylene glycol hydrogel.The number of COOH group is fixed by blutene (TBO) dyeing Amount.[24-25]Obtain the COOH group TBO at 633nm and absorb standard curve (Figure 14 A-14B).The density Estimation of COOH group It is 3.6 × 1016Molecule/cm2, this is than the COOH value obtained in the PEDOT-COOH/PEDOT of electrochemical deposition in naked gold surface (1.7×1016Molecule/cm2) the highest twice.
Use EDC-NHS, anti-cattle IFN-γ antibody covalency is fixed on carboxylation PEDOT in conducting polymer hydrogel On chain.Cyclic voltammetry is used to study the response of the conducting polymer hydrogel being fixed with antibody in PBS.This high connductivity Property hydrogel matrix in PBS, even have response, need not use outside oxidation-reduction indicator.It is fixed with leading of antibody The restructuring B-IFN-γ of the surface variable concentrations of electric polymer hydrogel excites, and records cyclic voltammetry curve.Containing antibody Gel is hindered electronics to the capture of B-IFN-γ and is transmitted by polymer chain, and causes oxidoreduction peak to decline (Figure 11 C). PEDOT reduction peak defines the base of this analyte electrochemical sensing in response to the decline of the B-IFN-γ (and generation) of variable concentrations Plinth.
We obtain the reduction current of the conductive hydrogel of antibody functionalization on restructuring B-IFN-γ concentration vs. electrode Standard curve (Figure 11 D).It is believed that the combination of target molecule and the PEDOT chain of antibody functionalization of electrochemistry insulation hinders Charge transport can which results in the suppression of reduction current with the electric conductivity of PEDOT.Additionally, carried out sandwich in ITO electrode Method is analyzed so that immobilized B-IFN-γ image conversion.Described electrode is hatched with restructuring B-IFN-γ subsequently, uses biotin afterwards The secondary antibodies changed and the Streptavidin of fluorogen coupling are hatched.Use fluorescence microscope detection fluorescence.Use fluorescein labelling Antibody incubation show restructuring B-IFN-γ and secure the combination (Figure 15 A: fluoroscopic image) of conductive hydrogel of antibody.
Embodiment 2: prepare sensor
As being previously reported by, use photolithography (photolithography) and wet etching method preparation gold and ITO Patterned electrodes.Micro-patterning sheet glass (including the photoresist at gold surface top) oxygen plasma processes 10 minutes, and In a nitrogen atmosphere, (3-acryloxypropyl) silicochloroform in toluene 0.05% solution in hatch about 1 little Time, to obtain the silane self assembled monolayer of glass region.This process is more particularly described below.
Positive corrosion-resisting agent (S1813) rotary coating (2000 turns, 30 seconds) is in coating by 15nm chromium adhesion layer and 100nm layer gold Sheet glass on, result in photoresist thick-layer.The 115 DEG C of softenings on hot plate of the sheet glass of photoresist coating toast 1 point Clock, then places and contacts with photomask, and be exposed under 365nm ultraviolet source.Described base plate is then placed in developer solution (MF319).After development step, the sheet glass of gold coating is dipped in gold etching solution, is dipped in subsequently in chromium etching solution.Never light The region of resistance material protection optionally removes metal, forms the golden micrographics of a diameter of 1500 μm.It is essential that after Shi Ke Remove the photoresist layer at gold microelectrode top the most at once, but use it for protecting bottom in silane-modified procedure Gold region, thus do not lose electric conductivity.
Base plate with the gold electrode of photoresist coating is modified with (3-acryloxypropyl) silicochloroform.Silane After modification, in acetone base plate is carried out supersound process to remove photoresist.
Then the nanoporous comprising PEDOT-polyethylene glycol hydrogel described in embodiment 1 is prepared in top of electrodes Film, and nano-porous films is attached on base plate.Then, also it is by the process of embodiment 1, bio-identification is partially attached to PEDOT-polyethylene glycol hydrogel.
Embodiment 3: pulmonary tuberculosis detects
The PEDOT conductive hydrogel of antibody (cattle-IFN-γ) functionalization is placed in the electrochemistry hole that embodiment 2 is arranged. In the target substance (recombinant interferon-gamma) of the variable concentrations of preparation introduces electrochemistry hole in 1X PBS, hatch about 20 minutes. Record cyclic voltammetry curve.
As shown in Figure 8 A, each induction electrode (conductive hydrogel of the antibody functionalization on gold microelectrode) is contacted by gold Pad is connected to manostat.By this way, each induction electrode addresses (addressed) by manostat respectively.
The gold electrode on sensing wafer with Signa Gel is working electrode, and is addressed by manostat respectively.We Employ outside Ag/AgCl reference electrode and platinum wire counter electrode.These are immersed in the electrochemistry hole arranged as shown in the figure respectively In electrolyte solution, and it is connected to manostat.
For the specificity of testing conductive hydrogel bioelectrode, the described sensor non-specific cell factor and albumen (including TGF-β, IL-6, TNF-α and IgG) excites.It is observed that for the non-specific cell factor (Figure 11 A) and people IFN- Signal minimum for γ (Figure 11 B) declines.This result shows, our sensor can detect that the non-specific cell factor mixes B-IFN-γ in thing.In order to prove our the sensor feasibility to cell related experiment, we want detection containing serum Signal response in culture medium and whole blood.As shown in Figure 11 C, compared with independent RPMI culture medium, add whole blood and cause and can neglect The loss of signal slightly.The conductive hydrogel sensor securing antibody excites with known B-IFN-γ, this B-IFN-γ (1:1) It is incorporated into without (Figure 16 A-16B) in the Sanguis Bovis seu Bubali/PBS of TB.The addition of Sanguis Bovis seu Bubali, compared with PBS, creates～the signal of 20% damages Lose, but, despite the loss of signal, after adding the B-IFN-γ of series concentration, change still detected.Use from Sanguis Bovis seu Bubali sample The Ox blood plasma sample of middle purification, we compare our conducting polymer hydrogel and ELISA method results, and it was found that Association between ELISA result and electrochemical signals (Figure 11 D).Real-time prison for the B-IFN-γ of release from Sanguis Bovis seu Bubali cell Surveying, conductive hydrogel cattle peripheral blood lymphocytes (PBMCs) securing antibody excites.Stimulate in mitogenesis Under, it was found that the change (Figure 11 E) of electrochemical source of current.As comparison, described electric current is having the cell of irriate, is lacking antibody Conductive hydrogel or be fixed with antibody, be not changed on the unprovoked conductive hydrogel of cell.Therefore, in our current research, We can confirm that our sensor has specificity and response to B-IFN-γ.
Although for the purpose that clearly understands, have been illustrated by describing some details of aforementioned invention with embodiment, but this Skilled person should be understood that some changes and modifications and can implement within the scope of the appended claims.Additionally, it is provided herein Each list of references is included in herein by reference of text, just looks like that every list of references is the same separately through quoting the degree included in. When between the application with list of references provided herein, existence conflicts, it is as the criterion with the application.

Claims (38)

1. a sensor, including:
Base plate；
At least one contacts the electrode of described base plate；
Cover the nano-porous films of described electrode；And
Bio-identification part selected from lower group: peptide, antibody, enzyme and fit, wherein said bio-identification some covalent is attached to institute State on electrode, or be covalently bound to the PEDOT random copolymer embedding in described nano-porous films, described PEDOT random copolymerization Thing has a Formulas I structure:
Wherein, each R is independently selected from lower group: OH and described bio-identification part, and at least one of which R is biological identification division, Further,
X and y independently be the integer from about 1 to about 1000, and wherein x and y sum is about the integer of 2 to about 1000.
2. sensor as claimed in claim 1, it is characterised in that described electrode is the gold electrode of pattern.
3. sensor as claimed in claim 1, it is characterised in that described sensor include central authorities' working electrode, cincture anti- Electrode and reference electrode.
4. sensor as claimed in claim 3, it is characterised in that described working electrode is gold electrode.
5. sensor as claimed in claim 3, it is characterised in that described reference electrode is silver/silver chloride electrode.
6. sensor as claimed in claim 1, it is characterised in that described bio-identification part is antibody.
7. sensor as claimed in claim 1, it is characterised in that described bio-identification part is fit.
8. sensor as claimed in claim 1, it is characterised in that described bio-identification some covalent is attached to be embedded in nanometer PEDOT random copolymer in perforated membrane, wherein, described nano-porous films includes having about 1000Da to about 10,000Da molecule The polyethylene glycol hydrogel of the PEG chain of amount.
9. sensor as claimed in claim 8, it is characterised in that described polyethylene glycol hydrogel includes having about 6000Da and divides The PEG chain of son amount.
10. sensor as claimed in claim 8, it is characterised in that described PEG-hydrogel is covalently attached to base plate.
11. sensors as claimed in claim 8, it is characterised in that the ratio of x Yu y is from about 10:1 to about 1:10.
12. sensors as claimed in claim 11, it is characterised in that the ratio of x Yu y is about 1:4.
13. sensors as claimed in claim 1, it is characterised in that farther include the trace for accommodating biosensor Titration orifice plate.
14. sensors as claimed in claim 1, it is characterised in that farther including port lid, described port lid comprises one or many The wall of the described base plate of individual contact, described wall defines the border, hole around electrode.
15. sensors as claimed in claim 1, it is characterised in that described nano-porous films is pellumina.
16. sensors as claimed in claim 15, it is characterised in that described bio-identification some covalent be connected to electrode with The self assembled monolayer of bio-identification part is formed on electrode.
17. sensors as claimed in claim 16, it is characterised in that described bio-identification part is for having oxidoreduction report Part is fit with thiol moiety, and wherein said thiol moiety is covalently attached to metal working electrode.
18. 1 kinds are used for the method detecting the disease marker in biological specimen, including by described in any one of claim 1-17 Sensor contacts with biological specimen, and detects the combination of described disease marker and bio-identification part, thus detects described disease Sick labelling.
19. methods as claimed in claim 18, it is characterised in that described detection disease marker and the combination of bio-identification part Including the peak value reduction current measuring PEDOT random copolymer.
20. methods as described in any one of claim 18-19, it is characterised in that utilize square wave voltammetry to detect described disease Labelling and the combination of bio-identification part.
21. methods as described in any one of claim 18-20, it is characterised in that described disease marker show by pulmonary tuberculosis or Hepatitis C infections.
22. methods as described in any one of claim 18-21, it is characterised in that described disease marker includes IFN-γ.
23. methods as claimed in claim 22, it is characterised in that described bio-identification part includes interferon-γ (IFN- γ) antibody.
24. methods as claimed in claim 22, it is characterised in that described bio-identification part includes being specific to the suitable of IFN-γ Body.
25. 1 kinds of conductive hydrogels, including:
PEG (PEG) hydrogel of covalent cross-linking；With
Embedding poly-(3,4-rthylene dioxythiophene) (PEDOT) random copolymer of described PEG-hydrogel, described PEDOT is randomly altogether Polymers has a Formulas I structure:
Wherein, each R is independently selected from lower group: OH and bio-identification part, and described bio-identification part is selected from lower group: peptide, anti- Body, enzyme and fit, at least one of which R is described bio-identification part, and
X and y independently be the integer from about 1 to about 1000, and wherein x and y sum is about the integer of 2 to about 1000.
26. conductive hydrogels as claimed in claim 25, it is characterised in that described polyethylene glycol hydrogel includes having from about The PEG chain of 1000Da to about 10,000Da molecular weight.
27. conductive hydrogels as claimed in claim 26, it is characterised in that described polyethylene glycol hydrogel includes having about The PEG chain of 6000Da molecular weight.
28. conductive hydrogels as claimed in claim 25, it is characterised in that the ratio of x Yu y is from about 10:1 to about 1:10.
29. conductive hydrogels as claimed in claim 28, it is characterised in that the ratio of x Yu y is about 1:4.
30. conductive hydrogels as claimed in claim 28, it is characterised in that the PEG hydrogel of described covalent cross-linking Prepared by Polyethylene Glycol-diacrylate.
31. 1 kinds of methods preparing conductive hydrogel as claimed in claim 25, including:
Under conditions of suitably forming polyethylene glycol hydrogel, Polyethylene Glycol-diacrylate is contacted with light trigger；
Be enough to be formed poly-(3,4-ethene dioxythiophene) (PEDOT) random copolymer of being embedded in described PEG-hydrogel Under electropolymerizatioconditions conditions, by described PEG-hydrogel with containing 3,4-rthylene dioxythiophene (EDOT) and 2,3-dihydro-thiophene also [3,4- B] the solution contact of [Isosorbide-5-Nitrae] dioxane-2-carboxylic acid (EDOT-COOH), described poly-(3,4-rthylene dioxythiophene) (PEDOT) is random Copolymer has a Formulas I:
Wherein, each R is OH, and x and y independently be the integer from about 1 to about 1000, and wherein x and y sum is about 2 to about The integer of 1000；Further,
Under conditions of being enough to described bio-identification some covalent is bound to described PEDOT random copolymer, by described water-setting Glue and bio-identification part contact, thus form the PEDOT random copolymer of Formulas I, at least one of which R is the life selected from lower group Thing identification division: peptide, antibody and fit, thus prepare the conductive hydrogel of claim 25.
32. 1 kinds of methods as claimed in claim 31, it is characterised in that described Polyethylene Glycol-diacrylate has from about The molecular weight of 1000Da to about 10,000Da.
33. methods as claimed in claim 32, it is characterised in that described Polyethylene Glycol-diacrylate has about 6000Da Molecular weight.
34. methods as claimed in claim 31, it is characterised in that the polymerizing condition of described formation polyethylene glycol hydrogel includes Irradiate Polyethylene Glycol-diacrylate.
35. methods as claimed in claim 31, it is characterised in that described EDOT and EDOT-COOH is with from about 10:1 to about 1: The ratio of 10 exists.
36. methods as claimed in claim 35, it is characterised in that described EDOT and EDOT-COOH deposits with the ratio of about 1:4 ?.
37. methods as claimed in claim 31, it is characterised in that described bio-identification part is antibody.
38. methods as claimed in claim 31, it is characterised in that described bio-identification part is fit.
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